Telomeres are repetitive nucleic acid sequences present at the ends of the linear chromosomes of eukaryotic organisms. The telomere sequences, together with telomere-binding proteins, confer stability to chromosomes. Telomeres are generally composed of short tandem repeats with a repeat sequence unit specified by the telomerase enzyme particular to the organism. Telomere repeat sequences are known for a variety of organisms. The human telomere repeat sequence unit is (TTAGGG)n. In addition to the double stranded repeat sequences, the 3′ ends of some telomeres contain a single-stranded region, which for humans is located on the G rich strand.
Telomerase is a riboprotein which synthesizes telomeric DNA. In the absence of telomerase, telomeres gradually shorten because DNA polymerases are unable to replicate the ends of linear duplex DNA. The gradual shortening of the telomeres ultimately leads to cell cycle arrest or cell death. In humans, telomere length dependent mortality in cells occurs because of telomerase repression in normal somatic cells before birth, an initial telomere length at birth and throughout life and a tightly regulated expression of telomerase in progenitor or stem cells. Humans are born with “full-length” telomeres. As telomerase is down-regulated in somatic tissues, this leads to loss of telomeric DNA with cellular and chronological age. Thus telomeres act as a mitotic clock, conferring a finite capacity for division on normal human cells. Short telomeres impair the ability of stem cells to proliferate. For example, short telomeres in epidermal stem cells impair skin and hair growth.
Tumor cells, which arise from normal cells, often have shorter telomeres than normal tissues but maintain their telomeres by expression of telomerase. There are three telomere dependent regulatory checkpoints on cell mortality. The first DNA damage checkpoint triggers replicative senescence when the mean telomere length is less than 5 kbp. The second checkpoint triggers cell death when the mean telomere length is reduced to 1-3 kbp. The third checkpoint called telomere uncapping can occur at any telomere length and is triggered by abnormally structured telomeres. (Harley Nature vol. 8 (March 2008)) Tumor cells undergoing rounds of mutation and clonal expansion during tumorigenesis rapidly deplete telomere length and will die if telomerase is not activated. During the progression of cancer, tumor cell populations evolve through sequential genetic and epigenetic changes allowing them to escape the normal somatic cellular controls. One of the tumor escape mechanisms is telomerase activation to circumvent the telomere dependent pathways of cell mortality. Telomerase is expressed in the majority of tumors from all cancer types. Thus tumors often have significantly shorter telomeres than normal tissues but with increased telomerase expression. (Harley et al., Nature, vol. 8, pp. 167-179, 2008).
Formalin-fixed, paraffin-embedded tissue is an invaluable resource of biological samples from medical procedures. Such tissues include, for example, cancer or tumor biopsies. These tissues are formalin-fixed and paraffin embedded after removal from the patient and archived for future reference. These archived tissues could be a useful source of information regarding the length of telomeres in the tumor tissue and the surrounding normal tissue. It would be useful to measure telomere length in the formalin-fixed paraffin-embedded tissue samples. However, a rapid and high throughput method of measuring the mean telomere length in formalin fixed paraffin embedded tissue has not yet been developed.
Some slower, laborious methods are available for determining telomere length in tissues. In one method, telomere length is determined by measuring the mean length of a terminal restriction fragment (TRF). The TRF is defined as the length, in general the average length, of fragments resulting from complete digestion of genomic DNA with restriction enzyme(s) that do not cleave the nucleic acid within the telomeric and subtelomeric sequences, and but do cleave frequently within single copy genomic sequences. The resulting terminal restriction fragment contains both telomeric repeats and subtelomeric DNA. The restriction digested genomic DNA is separated by electrophoresis and blotted onto a support, such as a nylon membrane. The fragments containing the telomere sequences are detected by hybridizing a probe specific for telomere sequences to the membrane. Upon visualization of the telomere containing fragments, the mean lengths of the terminal restriction fragments can be calculated. TRF estimation by Southern blotting gives a distribution of telomere length in the cells or tissue and thus the mean telomere length. However, the TRF assay is a laborious process and requires relatively large quantity of genomic DNA. In addition, it is not suitable for formalin-fixed, paraffin embedded (FFPE) samples because of the heavy DNA cross-linking and fragmentation resulting from formalin fixation of the tissue.
A quantitative polymerase chain reaction (Q-PCR) based method for relative telomere length measurement in normal fresh (unfixed) tissue samples was described Cawthon Nucleic Acids Research vol. 30, no. 10 (2002) and Cawthon, Nucleic Acids Research vol. 37, no. 3 (2009). The protocol utilizes two sets of primers. One set of primers is used to amplify telomere hexamer repeats. The other set of primers is used to amplify a single copy gene in the genome such as acidic ribosomal phosphoprotein (36B4). Telomere length is expressed as the telomere product normalized by single copy gene product. In other words, relative telomere length of a sample is the factor by which the experimental sample differs from a reference DNA sample in its ratio of telomere repeat copy number to single gene copy number. The quantity of telomere repeats in each experimental sample is measured as the level of dilution of an arbitrarily chosen reference DNA sample that would make the experimental and reference samples equivalent with regard to the number of cycles of PCR needed to generate a given amount of telomere PCR product during the exponential phase of PCR amplification. Similarly the relative quantity of the single copy gene in each experimental sample is expressed as the level of dilution of the reference DNA sample needed to match it to the experimental sample with regard to the number of cycles of PCR needed to generate a given amount of single copy gene PCR product during the exponential phase of the PCR. For each experimental sample, the ratio of these dilution factors is the relative telomere to single copy gene (T/S) ratio. Thus T/S=1 when the unknown DNA is identical to the reference DNA in its ratio of telomere repeat copy number to single copy number. The reference DNA sample (to which all of the experimental samples in a given study are compared) can be from a single individual or it can be a pooled sample from multiple individuals. The T/S ratio of one individual relative to the T/S ratio of the reference individual or the pooled sample corresponds to the relative telomere length of the DNA from the individual.
The assay has been adopted for telomere length measurement in freshly isolated leukocytes. However, it has not been successfully used for FFPE samples. (Koppelstaetter et al., Mechanisms of Ageing and Development vol. 126 1331 (2005)) The major technical challenges are DNA cross-linking and fragmentation, impurity in the isolated DNA, variable PCR amplification efficiency and normalization of telomere signal with a single copy gene signal in the same sample.
We describe here a quantitative PCR protocol that can reliably measure the telomere lengths in cells or tissues that have been formalin-fixed and paraffin embedded.